JP2011500118A - Methods for preparing porous scaffolds for tissue engineering, cell culture, and cell delivery - Google Patents

Abstract

  The present invention relates to a method for preparing a porous scaffold for tissue engineering. Another object of the present invention is to provide a porous scaffold obtained by the above method, and the use of the porous scaffold for tissue engineering, cell culture and cell delivery. The method of the present invention comprises the steps of: a) preparing an alkaline aqueous solution containing an amount of at least one polysaccharide, an amount of a cross-linking agent, and an amount of a pore-forming agent; and b) the solution is about 4 ° C to Converting the solution to a hydrogel by allowing the amount of polysaccharide to crosslink at about 80 ° C. for a sufficient amount of time; and c) immersing the hydrogel in an aqueous solution; and d) step c). And washing the porous scaffold obtained in (1).

Description

  The present invention relates to a method for preparing a porous scaffold for tissue engineering. Another object of the present invention is to provide a porous scaffold obtained by the above-described method and use of the porous scaffold for tissue engineering, cell culture and cell delivery.

  Tissue engineering is generally defined as creating tissue or organ equivalents by seeding cells on or within a scaffold suitable for transplantation. In order to form a tissue or organ equivalent on a scaffold, the scaffold must be biocompatible and cells must be able to attach and grow on the scaffold. These scaffolds can therefore be considered as substrates for the growth of cells in vitro or in vivo.

  The characteristics of an ideal biocompatible scaffold include the ability to support cell growth in vitro or in vivo, the ability to support the growth of various cell types or cell lines, and the varying degrees of flexibility required. Or the ability to have high rigidity, the ability to have various degrees of biodegradability, the ability to be introduced into the target site in vivo without inducing secondary injury, and the drug or bioactive substance to the desired site of action. The ability to be a vehicle or reservoir for delivery will be mentioned.

  Many different scaffold materials have been utilized for use in tissue regeneration guidance methods and / or as biocompatible surfaces. Biodegradable polymeric materials are often preferred because the scaffold is gradually degraded and eventually the cell-scaffold structure is completely replaced by cells. Many candidate materials that are said to aid in tissue growth or regeneration, which can be useful scaffolds, include gels, foams, sheets, and numerous porous particulate structures that differ in shape and shape. Can be mentioned.

  A wide variety of natural polymers disclosed to be useful for tissue engineering or tissue culture include fibronectin, each type of collagen, and laminin, and cells such as keratin, fibrin and fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate Various components of the outer matrix can be mentioned.

  Other commonly used polymers include poly (lactide-co-glycolide) (PLG). PLG is a mechanically strong, hydrolyzable polymer approved by the FDA for use in the body (Thomson RC, Yaszemski MJ, Powers JM, Mikos AG. Fabrication of biodegradable polymers ef entJ Biometer Sci Polyd Ed. 1995; 7 (1): 23-38; Wong WH.Mooney DJ.Synthesis and properties of biodegradable polymers used assynthetic mattress. J, editors; Langer R, Vacanti JP, associate editors. Synthetic biogradable polymer scaffolds. Boston: Birkhauser: 1997. p. 51-82.). However, PLG is hydrophobic and is generally processed under relatively stringent conditions, thus making factor incorporation and viable cell encapsulation potentially a challenge.

  As an alternative, various hydrogels, which are polymeric materials with a high moisture content (water content higher than 30% by weight), are used as scaffold materials. These are composed of synthetic or naturally derived hydrophilic polymer chains. The structural integrity of the hydrogel depends on the crosslinks formed between the polymer chains through various chemical bonds and physical interactions.

  For example, US Pat. No. 6,586,246 B1 discloses a method for preparing a porous hydrogel scaffold that can be used as a support for tissue engineering or culture substrates. The method of this document consists of: a) dissolving a biodegradable synthetic polymer in an organic solvent to prepare a highly viscous polymer solution; b) adding a pore-forming agent to the solution; and c) converting the polymer into a mold. Pouring; d) removing the organic solvent; e) immersing the polymer / salt gel slurry from which the organic solvent has been removed in a hot aqueous or acidic solution to cause salt foaming at room temperature to form a porous scaffold; Comprising the steps of: However, this method of preparing a porous hydrogel involves the use of an organic solvent with a synthetic polymer, which makes the method according to the invention less compatible for biological and therapeutic purposes.

  Accordingly, there is still a need in the art to develop a method for preparing porous scaffold matrices that can be used for biological and therapeutic purposes.

Therefore, the object of the present invention is to
a) preparing an alkaline aqueous solution comprising an amount of at least one polysaccharide, an amount of a cross-linking agent, and an amount of a pore-forming agent;
b) converting the solution to a hydrogel by placing the solution at a temperature of about 4 ° C. to 80 ° C. for a time sufficient for the amount of polysaccharide to cross-link;
c) immersing the hydrogel in an aqueous solution;
d) providing a method for preparing a porous scaffold, comprising the step of washing the porous scaffold obtained in step c).

  Another object of the present invention is to provide a porous scaffold obtainable by the above method.

  Yet another object of the present invention is to provide use of the porous scaffolds of the present invention for tissue engineering, cell culture, and cell delivery.

Definitions As used herein, the term “polysaccharide” refers to a molecule comprising two or more monosaccharide units.

  As used herein, the term “alkaline solution” refers to a solution having a pH above 7.

  As used herein, the term “acidic solution” refers to a solution having a pH below 7.

  As used herein, the term “aqueous solution” refers to a solution in which the solvent is water.

  The term “cross-linked” refers to a covalent linkage between one polymer chain and another polymer chain.

  The term “pore forming agent” means any solid agent that has the ability to form pores in a solid structure.

  As used herein, a “scaffold” is defined as a semi-solid system that includes a three-dimensional network of one or more polysaccharide chains. Such structures can contain varying amounts of water in equilibrium, depending on the characteristics of the polysaccharide (or polysaccharides) used and the nature and density of the network structure.

  The term “crosslinker” includes any agent that can introduce a crosslink between the chains of the polysaccharide of the invention.

  As used herein, the term “biodegradable” refers to a material that is degraded in vivo to a non-toxic compound that can be excreted or further metabolized.

Porous scaffold and its preparation method The first object of the present invention is to
a) preparing an alkaline aqueous solution comprising an amount of at least one polysaccharide, an amount of a covalent crosslinker, and an amount of a pore-forming agent;
b) converting the solution to a hydrogel by placing the solution at about 4 ° C. to about 80 ° C. for a time sufficient for the amount of polysaccharide to crosslink; and c) converting the hydrogel to an aqueous solution. Soaking step,
d) A method for preparing a porous scaffold comprising the steps of: washing the porous scaffold obtained in step c).

  Any kind of polysaccharide can be used in the present invention. Synthetic or natural polysaccharides may alternatively be used for the purposes of the present invention. For example, suitable natural polysaccharides include dextran, agar, alginic acid, hyaluronic acid, inulin, pullulan, heparin, fucoidan, chitosan, scleroglucan, curdlan, starch, cellulose, and mixtures thereof. It is not limited. Monosaccharides that can be used to produce the desired polysaccharide include, but are not limited to, ribose, glucose, mannose, galactose, fructose, sorbose, sorbitol, mannitol, iditol, dulcitol, and mixtures thereof. . For example, including chemically modified polysaccharides having acidic groups (carboxyl groups, sulfuric acid groups, phosphoric acid groups), amino groups (ethyleneamine, diethylamine, diethylaminoethylamine, propylamine), and hydrophobic groups (alkyl groups, benzyl groups) Good. Many of these compounds are commercially available from companies such as Sigma-Aldrich (St. Louis, Michigan, US).

  The preferred weight average molecular weight of the polysaccharide is from about 10,000 daltons to about 2,000,000 daltons, more preferably from about 10,000 daltons to about 500,000 daltons, and most preferably from about 10,000 daltons to About 200,000 daltons.

  In one embodiment of the invention, the polysaccharide (s) used to prepare the scaffolds of the invention are dextran, agar, pullulan, inulin, scleroglican, curdlan, starch, cellulose, or mixtures thereof. Such as neutral polysaccharides. In a preferred embodiment, a mixture of pullulan and dextran is used to prepare the scaffolds of the invention. For example, the mixture is composed of 25% dextran and 75% pullulan.

  In other embodiments of the invention, the polysaccharide (s) used to prepare the scaffolds of the invention are positively charged polysaccharides such as chitosan, DEAE-dextran, and mixtures thereof.

  In other embodiments of the invention, the polysaccharide (s) used to prepare the scaffolds of the invention are negatively charged polysaccharides such as alginic acid, hyaluronic acid, heparin, fucoidan, and mixtures thereof. .

  In another embodiment of the invention, the polysaccharide (s) used to prepare the scaffolds of the invention is a mixture of neutral polysaccharides and negatively charged polysaccharides, where the negatively charged polysaccharides Represents 1 to 20% of the mixture, preferably 5 to 10%.

In certain embodiments, the covalent crosslinker crosslinks trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl ₃ ), epichlorohydrin, formaldehyde, water soluble carbodiimide, glutaraldehyde, or polysaccharide. Selected from the group consisting of any other suitable compounds. In a preferred embodiment, the cross-linking agent is STMP. The concentration (w / v) of the covalent crosslinker in the aqueous solution is about 1% to about 6%, more preferably about 2% to about 6%, and most preferably about 2% to about 3%. It is. The recommended amount of crosslinking agent used is such that the weight ratio of polysaccharide to crosslinking agent is in the range of 20: 1 to 1: 1, preferably 15: 1 to 1: 1, more preferably 10: 1 to 1: 1. is there.

  Many of these compounds are commercially available from companies such as Sigma-Aldrich (St. Louis, Michigan, US).

  The aqueous solution containing the polysaccharide may further contain various additives depending on the intended use. Preferably, the additive is compatible with the polysaccharide and does not interfere with the effective cross-linking of the polysaccharide (s). The amount of additive used will depend on the particular application and can be readily determined by one skilled in the art using routine experimentation.

  The aqueous solution containing the polysaccharide may optionally contain at least one antimicrobial agent. Suitable antimicrobial preservatives are well known in the art. Examples of suitable antibacterial agents include alkylparabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, cresol, chlorocresol, hydroquinone, sodium benzoate, potassium benzoate, triclosan, and chlorhexidine. It is not limited to. Other examples of antibacterial and anti-infective agents that can be used include, but are not limited to, rifampicin, minocycline, chlorhexidine, silver ion agents, and silver-based compositions.

  The aqueous solution containing the polysaccharide may also optionally include at least one colorant to increase the visibility of the solution. Suitable colorants include dyes, pigments, and natural colorants. Examples of suitable colorants include, but are not limited to, alcian blue, fluorescein isothiocyanate (FITC), and FITC-dextran.

  The aqueous solution containing the polysaccharide may optionally also include at least one surfactant. As used herein, a surfactant refers to a compound that reduces the surface tension of water. The surfactant may be an ionic surfactant such as sodium lauryl sulfate or a neutral surfactant such as polyoxyethylene ether, polyoxyethylene ester, and polyoxyethylene sorbitan.

  In certain embodiments, the pore-forming agent may be an agent that can be converted to a gas under acidic conditions, and the pores are formed by carbon dioxide molecules that are leached from the polymer. Examples of such pore formers include, but are not limited to, ammonium carbonate, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, calcium carbonate, and mixtures thereof. The pore-forming agent is used in such an amount that the weight ratio of polysaccharide to pore-forming agent is in the range of 6: 1 to 1: 1, preferably 4: 1 to 1: 1, more preferably 2: 1 to 1: 1. It is preferable to do. Many of these compounds are commercially available from companies such as Sigma-Aldrich (St. Louis, Michigan, US). In some embodiments, the weight ratio of polysaccharide to pore former is 6: 1 to 0.5: 1, preferably 4: 1 to 0.5: 1, more preferably 2: 1 to 0.5: 1. It may be a range. In other embodiments, when the polysaccharide is a positively charged polysaccharide, the weight ratio of polysaccharide to pore former is 50: 1 to 1: 1, preferably 20: 1 to 1: 1, more preferably 10: It may be in the range of 1-1: 1.

  In this particular embodiment, the aqueous solution of step c) is an acidic solution. The acid may be selected from the group consisting of citric acid, hydrochloric acid, acetic acid, formic acid, tartaric acid, salicylic acid, benzoic acid, and glutamic acid.

  Alternatively, the pore former may be an inorganic salt that can dissolve when the cross-linked polysaccharide scaffold is immersed in water. An example of such a pore-forming agent is a saturated salt solution that will gradually dissolve. In this particular embodiment, the aqueous solution of step c) is an aqueous solution, preferably water, more preferably distilled water.

  Since the concentration of the pore forming agent affects the size of the pores formed in the scaffold, the size of the pores can be controlled by adjusting the concentration of the pore forming agent.

  The average pore size of the scaffold is about 1 μm to about 500 μm, preferably about 150 μm to about 350 μm, more preferably about 175 μm to about 300 μm. The density or porosity of the pores is about 4% to about 75%, preferably about 4% to about 50%.

  In another embodiment, the method of the invention can further comprise a step consisting of lyophilization of the scaffold obtained in step d). Freeze-drying can be performed using any apparatus known in the art. There are basically three types of freeze dryers: rotary evaporators, manifold freeze dryers, and shelf freeze dryers. Such devices are well known in the art and are commercially available such as freeze-dryer Lyovac (GT2, STERIS Rotary vane pump, BOC EDWARDS). Basically, the chamber vacuum is between 0.1 mBar and about 6.5 mBar. Freeze-drying is performed for a time sufficient to remove at least 98.5% of the moisture, preferably at least 99% of the moisture, more preferably at least 99.5% of the moisture.

  In other embodiments, the method of the present invention may further comprise a step consisting of hydrating a scaffold prepared according to the present invention. The water content is used to produce a scaffold having a desired moisture content in an aqueous solution (eg, deionized water, water filtered through a reverse osmosis membrane, physiological saline, or an aqueous solution containing an appropriate active ingredient). Perform by soaking for a sufficient time. For example, if a scaffold with a maximum moisture content is desired, the scaffold is immersed in an aqueous solution for a time sufficient for the scaffold to expand until its size or volume is maximized. Generally, the scaffold is immersed in the aqueous solution for at least about 1 hour, preferably at least about 2 hours, more preferably from about 4 hours to about 24 hours. Of course, the time required to hydrate the scaffold to the desired level depends on several factors such as the composition of the polysaccharide used, the size of the scaffold (eg, thickness), and the temperature of the aqueous solution, as well as other factors. Depends on.

  In certain embodiments, the moisture content of the hydrous scaffold is 80%, preferably 90%, more preferably 95%.

  In another specific embodiment, prior to step b), the aqueous solution of step a) can be poured into a mold so that the porous scaffold obtained using the method of the invention can be in the desired form. Any geometric mold may be used in accordance with the present invention. Different sizes may be assumed. For example, in general, an aqueous solution can be poured into a tubular mold having a central axis so that the porous scaffold has a tubular shape having a desired outer diameter and inner diameter. The mold may be prepared from any material, but preferred materials are those that have a non-stick surface, such as Teflon.

  Alternatively, the scaffold of the present invention may be cut and shaped to the desired size and shape.

  The method of the present invention may further comprise the step of sterilizing the scaffold using any suitable process. The scaffold can be sterilized at any suitable time, but is preferably sterilized before it is hydrated. Suitable radiation sterilization techniques include, for example, irradiation with cesium 137 at 35 gray for 10 minutes. Suitable non-radiation sterilization techniques include, but are not limited to, methods known in the art such as UV irradiation, gas plasma, or ethylene oxide. For example, the scaffold is sterilized using the sterilization system available from Abtox, Inc, Mundelein, IIIinois, under the trade name PlazLyte, or according to the gas plasma sterilization process disclosed in US Pat. Nos. 5,413,760 and 5,603,895. be able to.

  The scaffold produced by the method of the present invention can be packaged with any suitable packaging material. A packaging material that maintains the sterility of the scaffold until the packaging material is broken is desirable.

  In other embodiments, one or more biomolecules can be incorporated into the porous scaffold. In other embodiments, the biomolecules may include drugs, hormones, antibiotics, antimicrobials, dyes, radioactive substances, fluorescent substances, antibacterial substances, chemicals, or agents, any combination thereof. This material may be used for the purpose of increasing therapeutic effect, increasing visibility, proper orientation indication, resistance to infection, promoting healing, increasing flexibility, or any other desired effect. In said embodiment, the scaffold of the invention comprising one or more biomolecules as described above can be used as an active agent release control system.

  The scaffold produced by the method of the present invention is free of growth factors and other growth stimulators. In one embodiment, the biomolecule comprises chemotactic substances, antibiotics, steroidal or nonsteroidal analgesics, anti-inflammatory drugs, immunosuppressive drugs, anticancer drugs, various proteins (short peptides, bone forms). Cell-forming mediators; bioactive ligands; integrin binding sequences; ligands; various proliferative agents and / or differentiation inducers (epidermal growth factor, IGF-I, IGF) -II, TGF-β, growth and differentiation factors, stromal cell derived factor SDF-1; vascular endothelial growth factor, fibroblast growth factor, platelet derived growth factor, insulin derived growth factor, and transforming growth factor, vice Thyroid hormone, parathyroid hormone related peptide, bFGF; TGFβ superfamily factor; BMP-2; BM P-4; BMP-6; BMP-12; Sonic hedgehog; GDF5; GDF6; GDF8; PDGF, etc.); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate Fibronectin; decorin; thromboelastin; thrombin derived peptide; heparin binding domain; heparin; heparan sulfate; DNA fragment, DNA plasmid, Si-RNA, transfection reagent, or any combination thereof.

  In one embodiment, the growth factor is heparin binding growth factor (HBGF), transforming growth factor alpha or transforming growth factor beta (TGF beta), alpha fibroblast growth factor (FGF), epidermal growth factor (EGF). , Vascular endothelial growth factor (VEGF), and SDF-1, some of the same angiogenic factors. In other embodiments, factors include hormones such as insulin, glucagon, and estrogen. In certain embodiments, it may be desirable to incorporate factors such as nerve growth factor (NGF) or muscle morphogenic factor (MMF). In certain embodiments, TNFα / β, or matrix metalloproteinase (MMP) is incorporated.

  Furthermore, the scaffolds of the present invention optionally comprise anti-inflammatory agents such as indomethacin, acetylsalicylic acid, ibuprofen, sulindac, piroxicam, and naproxen; thrombogenic agents such as thrombin, fibrinogen, homocysteine, and estramustine; and sulfuric acid Radiation opaque compounds such as barium, gold particles, and iron oxide nanoparticles (USPIO) may be included.

  Furthermore, the scaffold of the present invention optionally selects an antithrombotic agent such as anti-vitamin K or aspirin, an antiplatelet agent such as aspirin, thienopyridine, dipyridamole, or clopidogrel (adenosine diphosphate (ADP) induced platelet aggregation) Or an anticoagulant such as heparin or fucoidan. A combination of heparin (anticoagulant) and tirofiban (antiplatelet agent) has been shown to be effective in reducing both thrombus and thromboembolism and can be incorporated into the scaffolds of the invention. Genistein that is a possible isoflavone with dose-dependent antiplatelet and antiproliferative properties and inhibits collagen-induced platelet aggregation responsible for primary thrombosis can also be incorporated into the scaffolds of the present invention.

Method of using the scaffold of the present invention The scaffold of the present invention is particularly suitable for tissue engineering, tissue repair, or tissue regeneration. Differences in porosity can facilitate the transfer of different cell types to the appropriate area of the scaffold. In other embodiments, the difference in porosity may facilitate the generation of appropriate intercellular linkages between the cell types that make up the scaffold, which are necessary for proper structuring of tissue development / repair / regeneration. For example, cell process extension can be adapted to be more appropriate through the various porosity of the scaffold material. Thus, the scaffold may comprise cells of any tissue.

  In certain embodiments, the scaffold is seeded with cells. In other embodiments, the scaffolds of the invention are immersed in a culture solution containing the desired cells for a time sufficient to allow the cells to penetrate the entire scaffold.

  In other embodiments, the scaffolds of the invention are capable of supporting the viability and proliferation of seeded cells in culture for an extended period of time without inducing differentiation.

  In other embodiments, the scaffolds of the invention provide a non-stimulatory cell growth environment (no activation by growth promoters).

  In other embodiments, the scaffolds of the invention are used to study physiological and pathological processes such as tissue growth, bone reconstruction, wound healing, tumor formation (including migration and invasion), differentiation and angiogenesis. be able to. Scaffolds allow the creation of a defined and controlled environment that can regulate and study specific processes in a controlled manner that does not include endogenous factors.

  Specifically, the scaffolds of the invention can be used for three-dimensional culture for diagnostic or toxic doses. In this embodiment, the scaffold of the present invention allows the product toxicity to be assessed directly against cells present in a three-dimensional environment. In said embodiment, the scaffold of the present invention is useful for evaluating the toxicity and / or pharmacological action of products such as hepatocytes, embryonic stem cells, epithelial cells, keratinocytes, or induced pluripotent stem cells (iPS cells). Used to culture fresh cells.

  In other embodiments, the scaffolds of the invention are capable of supporting the growth and differentiation of multiple cell types in vitro or in vivo.

  In other embodiments, the cells are stem cells or progenitor cells. In other embodiments, the cells are chondrocytes; fibrochondrocytes; bone cells; osteoblasts; osteoclasts; synovial cells; bone marrow cells; mesenchymal cells; epithelial cells, hepatocytes, muscle cells; Stem cells; embryonic stem cells; adipose tissue-derived progenitor cells; peripheral blood progenitor cells; stem cells isolated from adult tissues; induced pluripotent stem cells (iPS cells); gene transformed cells; chondrocytes and other cells A combination of bone cells and other cells; a combination of synovial cells and other cells; a combination of bone marrow cells and other cells; a combination of mesenchymal cells and other cells; a combination of stromal cells and other cells A combination of stem cells and other cells; a combination of embryonic stem cells and other cells; a combination of progenitor cells and other cells isolated from adult tissue; a combination of peripheral blood progenitor cells and other cells; a single cell from adult cells; Combination of detached stem cells and other cells; and gene transformation And a combination of cells and other cells, but is not limited thereto.

  In other embodiments, any of these cells for use in the scaffolds and methods of the invention can be, for example, a green fluorescent protein (GFP), a reporter gene (luciferase, alkaline, some of which are also angiogenic factors Phosphatase), heparin binding growth factor (HBGF), transforming growth factor α or transforming growth factor β (TGFβ), α fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and genetically modified to express desired molecules such as SDF-1. In other embodiments, expressed factors include hormones such as insulin, glucagon, and estrogen. In other embodiments, factors such as nerve growth factor (NGF) or muscle morphogenic factor (MMF) are expressed, or in other embodiments, TNFα / β is expressed.

  In certain embodiments, the scaffolds of the present invention can be prepared, for example, as described in Chaouat et al. (Chaouat M, Le Visage C, Authentier A, Chaubet F, Leturneur D. The evaluation of biosmatter-diameter. 2006 Nov; 27 (32): 5546-53.Epub 2006 Jul 20.) is suitable for preparing blood vessels for replacement of damaged arteries. Such surrogates can be prepared using the molds described above according to the method of the present invention. Such surrogates can then include cell populations to reconstruct blood vessels in vitro or in vivo. In other embodiments, the cells include, but are not limited to, mesenchymal stem cells (MSC), endothelial progenitor cells (EPC), endothelial cells, fibroblasts, and smooth muscle cells.

  In other specific embodiments, the scaffolds of the invention are suitable for preparing cartilage or bone implants. In such a case, the scaffold of the present invention can be loaded with chondrocytes, bone cells, osteoblasts, osteoclasts, vascular cells, or a mixture thereof, and cultured in the presence of a differentiation-inducing agent. Can do.

  The site of implantation depends on the affected / damaged tissue that needs treatment. For example, to treat articular cartilage, joint meniscus, and bone structural defects, a cell-seeded scaffold complex is placed at the defect site to facilitate repair of damaged tissue.

  In the case of central nervous system (CNS) damage, the scaffold complex may be seeded with a combination of adult neural stem cells, embryonic stem cells, glial cells, and Sertoli cells. In a preferred embodiment, the scaffold complex may be seeded with Sertoli cells from a heterologous or allogeneic source transformed cell line in combination with neural stem cells. Sertoli cells may be cultured with the scaffold complex until stem cells are added and then transplanted to the site of injury. This approach avoids one of the main obstacles to the application of cell therapy to the CNS, ie, the obstacle to stem cell survival after transplantation. A scaffold complex encapsulating a large number of Sertoli cells can provide a better environment for stem cell survival.

  Accordingly, porous polysaccharide scaffolds prepared according to the present invention include artificial blood vessels, artificial bladders, artificial esophagus, artificial nerves, artificial hearts, artificial heart valves, artificial skin, orthopedic implants, artificial muscles, artificial ligaments, artificial respiration. It can be effectively used as a raw material for producing an artificial tissue such as a vessel or an artificial organ. Furthermore, the porous polysaccharide scaffold of the present invention can be prepared in the form of a hybrid tissue by formulating or incorporating on or within other types of biomaterials and with functional cells from tissues or organs. . Hybrid tissues can have a variety of biomedical applications, for example, maintenance of cell functionality, tissue regeneration, and the like.

  Alternatively, the scaffolds of the invention can be used for cell delivery. Indeed, the scaffolds of the invention can be used as a raw material for preparing a cell delivery system that can be administered to a patient for therapeutic or diagnostic purposes. In certain embodiments, the scaffolds of the invention can be used to prepare patches, biological membranes, or dressings that can be carried by cells. For example, the scaffolds of the invention can be used to prepare dressings that can be applied to the skin to reconstruct or heal the skin. Alternatively, the dressing can be used for administration to a patient's heart to treat ischemia (myocardial infarction). Thus, in these embodiments, cells encapsulated in the scaffold can migrate into the targeted tissue or organ.

  In other embodiments, the scaffolds of the invention can be used for cell culture. The cells are then stimulated by adding appropriate growth factors to develop into differentiation or other physiological processes. A culture medium containing one or more cytokines, growth factors, hormones, or combinations thereof may be used to maintain the cells in an undifferentiated state or to differentiate the cells into a particular pathway.

  More specifically, the scaffold of the present invention can be used for generation of a target molecule. Indeed, the scaffolds of the invention can be used to provide a biological environment for fixing cells in a bioreactor so that the cells can produce the desired molecules. The scaffold of the present invention protects cultured cells mechanically and biochemically.

  Thus, the scaffold serves as a cell reservoir for generating desired molecules such as proteins, organic molecules, and nucleotides. For example, the protein of interest includes, but is not limited to, growth factors, hormones, signal molecules, cell growth inhibitors, and antibodies. The scaffolds of the invention are of particular interest for the production of monoclonal antibodies. The scaffolds of the present invention may also be suitable for the production of organic molecules such as fragrances, therapeutic molecules.

  For this purpose, the scaffold of the present invention may carry any cell type including prokaryotic and eukaryotic cells. For example, the scaffold of the present invention may carry bacteria, yeast cells, mammalian cells, insect cells, plant cells and the like. As a specific example, E.I. E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (eg, Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (eg, lymphatic cells) Blasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). More specifically, the present invention contemplates the use of established cell lines such as hybrid cells. Alternatively, the cells may be genetically modified to express the desired molecule.

  The scaffold of the present invention can be loaded with cells and cultured for a period of time, and then the cells can be recovered / extracted / separated from the scaffold and further used for therapeutic or diagnostic applications, or for cell analysis. Separation of cells from the scaffold may involve the use of an enzyme capable of degrading the scaffold such as pullulanase and / or the use of an enzyme capable of detaching cells such as collagenase, elastase or trypsin, or a cell detachment solution such as EDTA.

  The invention is further illustrated in the light of the following figures and examples.

It is a photograph of the porous scaffold obtained in Example 1 (scale: 6 mm). Porous scaffold obtained in Example 1 is a photograph of the scaffold analyzed by a scanning electron microscope (right side of image, scale: 200 micron). It is a graph which shows the light absorbency (570 nm) of formazan in the 1st day according to the initial cell number seed | inoculated on the porous scaffold.

<Example 1>
Preparation of polysaccharide-based scaffolds A polysaccharide-based scaffold was prepared using a mixture of pullulan / dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc. Okayama, Japan; Dextran, MW 500,000, Pharmacia). A polysaccharide solution was prepared by dissolving 9 g of pullulan and 3 g of dextran in 40 mL of distilled water. Sodium carbonate (8 g) was then added to the polysaccharide solution and stirring was continued until a homogeneous mixture was obtained. The polysaccharide was chemically cross-linked using the cross-linking agent trisodium trimetaphosphate STMP (Sigma, St Louis) under alkaline conditions. Briefly, 1 ml of 10M sodium hydroxide was added to 10 g of the polysaccharide solution, followed by 1 ml of water containing 300 mg of STMP. The mixture was then poured into a Petri dish (Nunclon®, # 150288) and incubated at 50 ° C. for 15 minutes. The resulting hydrogel was immediately immersed in a large beaker containing 20% acetic acid solution and left for at least 30 minutes. The resulting scaffold was extensively washed with pH 7.4 phosphate buffered saline followed by distilled water for at least 2 days. After the lyophilization step, the porous scaffold was stored at room temperature until use. Scanning electron microscope analysis confirmed the porosity of the scaffold (FIGS. 1 and 2).

<Example 2>
Types of polysaccharides Porous scaffolds were prepared as described in Example 1 using different types of polysaccharides at different ratios and keeping the total amount of polysaccharides at a constant value. The polysaccharide was either pullulan, dextran 500, fucoidan LMW (low molecular weight) and fucoidan HMW (high molecular weight).

  Solubilization (+++ indicates that the polysaccharide was completely solubilized) and the viscosity of the resulting polysaccharide solution (++ indicates that the solution is very viscous) were assessed visually. In all cases, porous scaffolds were obtained at the end of the protocol.

<Example 3>
Amount of Pore Forming Agent A porous scaffold was prepared as described in Example 1 with varying amounts of pore forming agent. Briefly, 2 g, 4 g, or 8 g of sodium carbonate was added to the pullulan / dextran solution.

  Visual assessment of solubilization (++ indicates that the polysaccharide is completely solubilized) and viscosity of the resulting polysaccharide solution (++ indicates that the solution is very viscous) and porosity did. For scaffolds prepared with the least amount of pore former (2 g), the foaming process was mild compared to the foam obtained with 4 g and 8 g pore former. In all cases, porous scaffolds were obtained at the end of the protocol.

<Example 4>
Crosslinker concentration Porous scaffolds were prepared as described in Example 1 with the amount of crosslinker varied from 200 mg to 500 mg.

  Visual assessment of solubilization (++ indicates that the polysaccharide has been completely solubilized) and viscosity of the resulting polysaccharide solution (++ indicates that the solution is very viscous) and porosity did. In all cases, porous scaffolds were obtained at the end of the protocol.

<Example 5>
Cell support of porous scaffold Human bone marrow mesenchymal stem cells (hMSC) were cultured on the scaffold prepared as in Example 1. A circular porous scaffold having a diameter of 6 mm and a thickness of 1 mm was cut out using a circular punch. The medium used was low glucose DMEM (Gibco, Life Technology, New York) containing 10% fetal bovine serum and 1% penicillin / streptomycin (Sigma). Cells were trypsinized and then rehydrated into a dry scaffold using 20 μL of cell suspension (10 ⁶ cells per scaffold). Samples were then maintained in 1 ml of medium for up to 1 week. Non-cell seeded porous scaffolds were incubated in medium and used as controls.

  Metabolic assays (MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) were performed to assess cell viability. Briefly, a stock solution of MTT (Sigma) 5 mg / mL was mixed 1:10 with DMEM. The scaffold was incubated with 1 mL of reagent solution at 37 ° C. for 3 hours. After washing the scaffold with PBS, the formazan crystals were solubilized in 0.3 mL of isopropanol / 0.04M HCl. Absorbance at 590 nm was recorded with a microplate reader (Multiskan, Thermo Electron Corporation, Waltham, Mass.). The absorbance at day 1 was directly proportional to the initial number of cells seeded on the scaffold (FIG. 3).

  Similar experiments were successfully performed using other cell types such as primary vascular smooth muscle cells and endothelial cells of animal and human origin.

<Example 6>
Confocal analysis of cell behavior in porous scaffolds Fluorescent scaffolds were prepared as in Example 1 by adding a small amount (5 mg) of FITC-dextran to the polysaccharide solution. HMSCs labeled according to the manufacturer's instructions using fluorescent markers (PKH26, SIGMA P9691) were seeded on fluorescent scaffolds as in Example 5. The porous structure of the scaffold was confirmed by confocal imaging.

<Example 7>
Cell viability by life-and-death assay Using confocal imaging, two fluorescent probes to measure cell membrane permeability, a cell-permeable green fluorescent dye (calcein AM) to stain live cells, and a non-cell to stain dead cells Based on the use of a permeable red fluorescent dye (propidium iodide), cell viability was assessed by a live / dead assay (Calbiochem, San Diego, Calif.). On day 7, only a few dead cells were found in the scaffold, with most cells being live cells.

<Example 8>
Effect of pore former on the porosity of the scaffold Porous scaffolds were prepared as described in Example 1 with varying amounts and properties of the pore former. For confocal analysis of the fluorescent porous scaffold, 5 mg of FITC-dextran was added to the polysaccharide solution. Optical sections were obtained using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 10 × Plan-NeoFluor objective (0.3 numerical aperture) (Carl Zeiss). FITC-dextran was excited at 488 nm using an argon laser and the fluorescence emission was selected by a 505-530 nm bandpass filter. The pore size was evaluated with ImageJ® software. The void volume was calculated using the Amira® software statistics / capacitance module, and the results were expressed as% volume of the scaffold.

<Example 9>
Positively charged polysaccharide DEAE-dextran was used as the only polysaccharide to prepare a porous scaffold with positive charge. Briefly, a DEAE-dextran solution was prepared by dissolving 1 g DEAE-dextran (Fluka reference # 30461) in 1.5 mL distilled water. Sodium carbonate (100 mg) was then added to the polysaccharide solution and stirring was continued until a homogeneous mixture was obtained. The polysaccharide was chemically cross-linked using the cross-linking agent trisodium trimetaphosphate STMP (Sigma, St Louis) under alkaline conditions. Briefly, 150 μL of 10M sodium hydroxide was added to the polysaccharide solution, followed by 150 μL of water containing 45 mg of STMP. The mixture was then poured into Petri dishes (Nunclon®, # 150288) and incubated at 50 ° C. for 15 minutes. The resulting hydrogel was immediately immersed in a large beaker containing 20% acetic acid solution and left for at least 30 minutes. The resulting scaffold was extensively washed with pH 7.4 phosphate buffered saline followed by distilled water for at least 2 days. After the lyophilization step, a porous scaffold was obtained and stored at room temperature until use.

<Example 10>
A negatively charged porous scaffold was prepared by adding a negatively charged polysaccharide fucoidan (Sigma reference # F5631) to the pullulan / dextran mixture. Briefly, a polysaccharide solution was prepared by dissolving 9 g pullulan and 3 g dextran in 40 mL distilled water, and then 1.2 g fucoidan was added to the polysaccharide solution. Next, sodium carbonate (8 g) was added to the polysaccharide solution, and a cross-linking process was performed as in Example 1 to obtain a three-dimensional scaffold containing a negatively charged polysaccharide.

<Example 11>
Differentiation of human mesenchymal stem cells into chondrocyte-like cells in a three-dimensional scaffold Human bone marrow mesenchymal stem cells (hMSCs) were cultured in scaffolds prepared as in Example 1 in a serum-free medium for chondrocyte formation. . The medium for chondrocyte formation was 10 ng / ml TGF-β3 (Oncogene, Cambridge, MA), 100 nM dexamethasone (Sigma, St Louis, MO), 170 μM ascorbic acid-2-phosphate (Sigma, St Louis, MO). ), And DMEM supplemented with 5 mL of ITS-Plus (Collaborative Biomedical Products, Bedford, Mass.). After 3 weeks of culture, the cell-seeded scaffold was fixed with 10% formaldehyde and then cryosectioned. Cryosections were stained with either 0.05% (w / v) toluidine blue or 0.1% safranin O solution. A strong positive staining for extracellular matrix synthesis is observed, indicating differentiation of MSCs into chondrocytes.

<Example 12>
Three-dimensional culture of hepatocytes HepG2 cells, human hepatocellular carcinoma cells in low glucose DMEM (Gibco, Life Technology, New York, USA) containing 10% fetal bovine serum and 1% penicillin / streptomycin (Sigma), Cultivated on a scaffold prepared as in Example 1. A circular porous scaffold having a diameter of 6 mm and a thickness of 1 mm was cut out using a circular punch.

  Cells were trypsinized and rehydrated into dry scaffolds using 20 μL of cell suspension (85,000 cells per scaffold). Samples were then maintained in 1 mL of medium for up to 1 week. Non-cell seeded porous scaffolds were incubated in medium and used as controls. Formation of hepatocyte spheroids was observed after 4 days of culture. Using calcein AM (Calbiochem, San Diego CA, USA), a polyanionic dye that is hydrolyzed by viable cells and thereby produces intense uniform green fluorescence (wavelength 485-535 nm) according to the manufacturer's instructions. The cell viability within the spheroids was assayed. Cell seeded scaffolds contained viable hepatocytes suitable for toxicology assays.

Claims (19)

a) preparing an alkaline aqueous solution comprising an amount of at least one polysaccharide, an amount of a covalent crosslinker, and an amount of a pore-forming agent;
b) converting the solution to a hydrogel by placing the solution at about 4 ° C. to about 80 ° C. for a time sufficient for the amount of polysaccharide to cross-link;
c) immersing the hydrogel in an aqueous solution;
d) A method for preparing a porous scaffold, comprising the step of washing the porous scaffold obtained in step c).   The method of claim 1, wherein the polysaccharide is selected from the group consisting of dextran, agar, alginic acid, hyaluronic acid, pullulan, inulin, heparin, fucoidan, chitosan, and mixtures thereof. The covalent cross-linking agent, trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl _3), epichlorohydrin, formaldehyde, is selected from the group consisting of water-soluble carbodiimide and glutaraldehyde, according to claim 1, or 2. The method according to 2.   The pore-forming agent is selected from the group consisting of ammonium carbonate, ammonium bicarbonate, calcium carbonate, sodium carbonate, and sodium bicarbonate and mixtures thereof, and the liquid of step b) is an acidic solution. The method according to any one.   The method according to any of claims 1 to 4, wherein the weight ratio of polysaccharide to pore former is in the range of 6: 1 to 1: 1.   The method according to any one of claims 1 to 5, wherein the weight ratio of polysaccharide to crosslinking agent is in the range of 15: 1 to 1: 1.   7. A method according to any one of the preceding claims, wherein the solution of step a) is poured into a mold before step b).   The method according to claim 1, wherein the scaffold is shaped.   The porous scaffold obtained by the method as described in any one of Claim 1 to 8.   The porous scaffold according to claim 9, wherein the pore size is 1 μm to 500 μm.   The porous scaffold according to claim 9 or 10, wherein the porosity ranges from 4% to 50%.   12. The porous scaffold according to any one of claims 9 to 11, which carries a certain amount of cells.   The porous scaffold of claim 12, wherein the cells are selected from the group consisting of yeast cells, mammalian cells, insect cells, and plant cells.   Mammalian cells are chondrocytes; fibrochondrocytes; bone cells; osteoblasts; osteoclasts; synovial cells; bone marrow cells; epithelial cells, hepatocytes, mesenchymal cells; stromal cells; muscle cells, stem cells; embryos 14. The porous scaffold of claim 13, selected from the group consisting of sex stem cells; adipose tissue-derived precursor cells; peripheral blood progenitor cells; stem cells isolated from adult tissue; and gene transformed cells.   15. A porous scaffold according to any one of claims 9 to 14 for tissue engineering, cell culture and cell delivery.   A substitute blood vessel produced using the scaffold according to any one of claims 9 to 12.   A cartilage or bone implant made using the scaffold according to any one of claims 9-12.   Use of the scaffold according to any one of claims 9 to 12 for product toxicity evaluation and / or pharmacological evaluation.   A controlled release system of an active agent, produced using the scaffold according to any one of claims 9 to 12.

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